Project Summary The long term goal of this research is to elucidate the biochemical processes that create cancer- causing mutations. Mutations in cancer tissues are composed of more than 30 patterns of mutations (cancer mutation signatures) that are likely related to cancer etiology. Despite their importance, most cancer mutation signatures have not been explained in biochemical detail. This project will biochemically reconstitute mutational processes that are induced by various DNA damaging agents and determine the detailed mutation profiles that are created during the processes. Comparison of the in vitro mutation profiles with the known cancer mutation signatures will demonstrate the specific biochemical processes that produce the cancer signatures. To quantify mutations, this project will use a newly developed NGS-based cell-free mutation assay system, in which randomly damaged DNA is incubated with various DNA polymerases, including a replicative polymerase Pol? and translesion polymerases Pol?, Pol?, Pol?, Pol?, and Rev1. Specific Aim 1 will determine the mutation profiles induced by various DNA damaging agents and investigate the molecular details of the mutational processes. The agents include: a) Agents that modify DNA directly: Ultraviolet light (UV), reactive oxygen species, acetaldehyde, b) Chemical carcinogens that (may) require metabolic activation: Benzo[a]pyrene and temozolomide, c) Spontaneous DNA damages that are accelerated by heat: Deamination of cytosine that converts cytosine to uracil, 5-methylcytosine to thymine. Specific Aim 2 will develop a new cell-free method to identify mutagens in crude compounds and describe their mutation profiles. This method is a combination of the cell-free mutation assay system and a LC-MS/MS. The ability of the new method will be tested by analyzing tobacco smoke extract, which contains many known carcinogens. Analysis of the smoke extract will be a good benchmark to test the capability (how many known mutagens can be detected) of the proposed method. In addition, positive results will reveal the mutation profiles of multiple mutagens in tobacco smoke. The new method may fill an important technical gap in current screening methods of carcinogens. The outcome of this project will be important to understand the etiology of cancer-causing mutations, and hopefully to find a way to prevent cancers.